Synthesis of Novel Peptides Using Unusual Amino Acids

Small peptides are valuable peptides due to their extended biological activities. Their activities could be categorized according to their low antigenicity, osmotic pressure, and also because of their astonishing bioactivities. For example, the aggression of Phe-Phe fibers via self-assembly and intermolecular hydrogen bonding is the main reason for the formation of Alzheimer’s β-amyloid fibrils. Hydrogen bonding is the main intramolecular interaction in peptides, while the presence of aromatic ring leads to the π-π stacking and affects the self-assembly and aggression. Thus, insertion of an unusual amino acid into peptide sequence facilitates the formation of intramolecular bonds, lipophilicity and its conformation. To design new small peptides with remarkable lipophilicity, it is an idea to employ γ-amino acid, such as gabapentin (H2N-Gpn-OMe) and baclofen (H2N-Baclofen-OMe), in the structure of small peptides to increase cell-penetrating properties and to prevent aggression of Phe-Phe fibrils in β-amyloids of Alzheimer’s disease. Some new tri- and tetrapeptides were synthesized through introducing biologically active gabapentin and baclofen to dipeptide of phenylalanine (Phe-Phe) through solution phase peptide synthesis strategy.


Introduction
During digestion or degeneration of proteins, small peptides are formed that are of vital sources of nutrition for human and animals (1). Particularly, di-and tri-peptides are the most valuable ones due to the low antigenicity, osmotic pressure, and also because of their astonishing bioactivities, e.g. antioxidative, antimicrobial, antihypertension, and immunomodulatory (2,3).
Beside the special biological activities of dipeptides, another surprising feature of them was discovered by Reches and Gazit in 2003. They found that L-Phe-L-Phe makes nanotubes in hexafluoro-2-propanol and water as solvents (4). Currently, the focus on the aggression of Phe-Phe fibers is because of their potential characteristic in the aggression of Alzheimer's β-amyloid fibrils. It has been found that the π-π stacking among phenyl rings of -F19-F20-is the main reason of amyloids aggression (5)(6)(7). Therefore, finding the properties of such fibers opens doors to discover new methods of treating various nervous diseases.
Hydrogen bonding is the main intramolecular interaction in peptides, while the presence of aromatic ring leads to the π-π stacking and affects the self-assembly and aggression. Thus, insertion of an unusual amino acid and sugars into peptide sequence increases the formation of intramolecular bonds, lipophilicity, and its conformation (8).
Modification of the backbone of α-peptides can result in proteolytically stable sequences; one of the important properties in the design of analogues of biologically active sequences (9). Moreover, modification of peptides with glycosyl and other types of highly soluble amino acids moieties has gained prominence because of awesome functional characteristics of these biomolecules (10)(11)(12). For instance, glycoconjugation of lysine residues using sugar vinyl sulfoxide led to an antimicrobial compound that catalyzes digestion of Gramnegative E. coli cell wall (13). Recently, aspartic thioacid-containing peptides were synthesized and easily converted to N-glycopeptides through a chemoselective thioacid-glycosylamine ligation (14). Asparagine containing glycopeptides linked to various saccharides were also prepared to develop the synthetic method of such valuable compounds (15).
Gabapentin (Gpn) is an available antiepileptic drug which is also used as a medicine for neuropathic pains (16). Due to the presence of cyclohexyl group in Gpn structure, construction of peptides with Gpn residues could affect conformation and lipophilicity of the final peptide (17). Racemate baclofen is a lipophilic analogue of GABA which acts as a muscle relaxer and an antispastic agent (18). Existence of 4-chlorophenyl moiety in its structure increases its lipophilicity. Constructing peptides with highly lipophilicity may affect their permanently remaining into the central nervous system (19). In order to peptide design, Gpn may be employed as a stereochemically constrained equivalent of its parent unsubstituted γ-aminobutyric acid residue (20). Hence, designing new small peptides fibres with remarkable lipophilicity is a good idea to increase cell-penetrating properties and to prevent aggression of Phe-Phe fibrils in β-amyloids of Alzheimer's disease. Alezra's research team found that small peptides including γ-amino acids may act as turn inducer to either form stable structures or enhance bioactivity of the molecules (21)(22)(23). One of them is probably gelation that has been observed in active γ-peptides (24). Such properties have been found as new advantages of self-assembled peptides for medicine (25).
Due to increasing interest for the synthesis of low molecular weight peptides, primarily, we were encouraged to design and synthesis some novel small peptides; to value this desire, the target small peptides were designed to enrich with biologically active γ-amino acid (Gpn and baclofen) and Phe-Phe dipeptide in their backbones. Correspondingly, the

General
All solvents were purchased as reagent grade, dried, using standard conditions and stored over molecular sieves. NMR spectra were carried out on a Bruker Avance (DRX-300 or DR-X 500 MHz) spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to residual solvent as an internal reference. The following abbreviations were used to explain the multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; dd doublet of doublets; m, multiplet; br, broad. The structure of all products was characterized by 1 H NMR (300 MHz) and thin layer chromatography (TLC) on silica gel and used without further purification. The purified final compounds were fully characterized by IR spectroscopy, 1 H NMR spectroscopy, and HR-mass spectrometry. Melting points were obtained on an Electrothermal 9100 capillary melting point apparatus. High-resolution mass spectra (HRMS) were performed on an Apex Qe-FTICR mass spectrometer. The IR spectra were obtained on a FT-IR ABB (FTLA 2000) spectrometer in liquid film and KBr pellets.

General procedure for the synthesis of di-, tripeptide with protected C-Terminal and N-Terminal (Peptide Coupling):
Boc-AA-OH (1 mmol), TBTU (1.1 mmol), HOBt.H 2 O and ethyl acetate (7 mL) were stirred for 10 min. Then, H-AA-OMe (1.2 mmol) and diisopropylethylamine (DIPEA) (3 mmol) were added and the mixture was stirred for 12 h. The progress of reaction was monitored by TLC (H 2 O/Methanol/ethyl acetate 1:2:10). The product was taken in ethyl acetate (60 mL). The aqueous phase was extracted with ethyl acetate and this operation was done repeatedly. The organic layer was washed with Na 2 CO 3 3% (3 × 50 mL), brine (2 × 50 mL), and acidified with a dilute solution of citric acid 20 % (3 × 50 mL), and brine (2 × 50 mL), then dried over anhydrous sodium sulfate and filtered and concentrated by rotary evaporator to get the product.

General procedure for deprotection of C-Terminus
To Boc-AA-AA-OMe (1 mmol), a mixture of MeOH (25 mL) and NaOH (4 mL, 2 M) was added and the progress of saponification was monitored by thin layer chromatography (TLC). The reaction mixture was stirred. After 10h, MeOH was evaporated under vacuum, the residue was taken in EtOAc (50 mL), washed with water (2 × 50 mL); acidity of the aqueous layer was adjusted at pH = 2 using citric acid 20% and it was extracted with EtOAc (3 × 50 mL). The extracts were pooled, dried over anhydrous Na 2 SO 4 and evaporated in vacuum.

General procedure for final Deprotection
To N-protected compound (1 mmol), triethylsilan (3 mmol) and a mixture of anhydrous TFA and DCM (1:1 v/v) was gradually added and stirred well. The progress of reaction was monitored by TLC (H 2 O/ MeOH/EtOAc 1:2:10). Then, excess solvent was evaporated under reduced pressure. The residue was purified by dissolving it in CH 2 Cl 2 and recrystallized by adding Et 2 O. Then, the product was collected on a filter, washed with Et 2 O, and dried at 50 °C in vacuum.
And since zwitterions have minimal solubility at their isoelectric point, final product was isolated by precipitating it from water by adjusting the pH to its particular isoelectric point. Then, product was collected on a filter, and dried at 50 ˚C in vacuum.  (2) 13 3) ppm. 13

Results and Discussion
To improve the functionality of F-F dipeptide, two γ-aminobutyric acids, Gpn, and Baclofen (Scheme 1) were introduced to its structure to increase the π-π stacking, and thus lipophilicity properties. Gabapentin is an anticonvulsant medication and its properties is owing to the presence of lipophilic cyclohexyl in its structure penetrating into the bloodbrain barrier and central nervous system (26). Besides, Baclofen includes 4-chlorophenyl ring which its presence in tri-peptide structure may increase the π-π stacking.
To synthesize tripeptides, two strategies were employed. The first one was relied on the core of Phe-Phe to introduce the third amino acid. The second one, Phenylalanine was bound to the C-terminus of γ-aminobutyric acids. In both strategies, solution phase peptide synthesis strategy was used (27).
To start the first strategy, protection reactions were essential for the preparation of amino acids, which include: carboxylic acid protection of Boc-Phe-OH through a methylation process using thionyl chloride in MeOH, and amine protection of Phe by di-tert-butyl dicarbonate (Boc 2 O). As shown in Scheme 2, the reaction steps involved: a) coupling reaction of protected amino acids which are Boc-Phe-OH and H-Phe-OMe using TBTU/HOBT as the coupling reagent, b) basic hydrolysis of methyl ester using

Conclusion
In conclusion, the synthesis of novel series of tri-and tetrapeptides were described and confirmed by inserting γ-amino acids of Gpn and Baclofen into the structure of Phe-Phe dipeptide. Adding the bioactive γ-amino acids to the Phe-Phe sequence could affect the lipophilicity and self-assembly of the peptides. The increased lipophilic properties of tri-and tetrapeptides leading to the nanostructural formation may affect their permeability onto the nervous system. The research to find the gel formation condition and also their activity is in progress in our lab.